Identification of metastasis associated gene G3BP by differential display in human cancer cell sublines with different metastatic potentials G3BP as highly expressed in non-metastatic

OBJECTIVE: To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression in two cell sublines derived from human giant cell carcinoma of lung (PG) which had different metastatic potentials. METHODS: Using in vivo tumorigenicity and a spontaneous metastasis assay in nude mice, two sublines (BE1, LH7) from human giant cell carcinoma of lung (PG) with different metastatic potentials were isolated and characterized. mRNA differential display was used to compare the levels of gene expression between them and the obtained results were confirmed by Northern hybridization. RESULTS: One differentially expressed band was nearly identical (99% homology) to Ras-GTPase-Activating protein SH3 domain binding protein (G3BP). G3BP displayed a strong expression in LH7 (non-metastatic in recipient nude mice) and a very weak expression in BE1 (100% metastatic frequency). The same different expression level of G3BP was detected in Northern hybridization with another panel of cell sublines with different metastatic potentials (established in our lab) derived from human prostate carcinoma cell line PC-3M. CONCLUSION: Our results indicate that G3BP was implicated in cancer metastasis because of its differential expressions in the two panels of cell sublines with different metastatic potentials.

Hepatic differentiation from embryonic stem cells in vitro

OBJECTIVE: To investigate an method for hepatic differentiation from embryonic stem cells (ES cells) in vitro and the resulting differentiation ratio, in order to develop a procedure for producing a new type of hepatocyte for hepatocyte replacement therapy in the treatment of liver failure. METHODS: ES cells from Balb/C mice were cultured and maintained in an undifferentiated state in gelatin-coated dishes using Dulbecco's modified Eagle's medium (DMEM) containing 1000 U/ml leukemia inhibitory factor (LIF). Then, LIF was withdrawn from the DMEM to allow the ES cells to develop into embryonic bodies (EBs). EBs were plated onto tissue culture dishes, and growth factors such as acidicfibroblast growth factor (aFGF) and hepatocyte growth factor (HGF) were added to the medium to promote directional differentiation. The course of development and differentiation was observed dynamically using an inversion microscope. The expression of hepatic proteins, such as alpha-fetoprotein (AFP), albumin (ALB), cytokeratin 8 (CK8), cytokeratin 18 (CK18), in cytoplasm was analyzed by immunocytochemistry (ICC). The concentration of ALB in the medium was determined dynamically by radioimmunoassay (RIA). RESULTS: ES cells replicated as clones, without differentiating, in DMEM containing LIF. They developed into EBs in medium without LIF. Our ICC assay showed that differentiating cells did not express hepatic proteins, such as AFP, ALB, CK8, and CK18 until day 7, day 9, day 11, and day 11, respectively (up to 2 days later when growth factors are not present). The concentration of AFP in the medium was first detected on day 8, at a concentration of 3.4 ng/ml, and increased to 22.8 ng/ml by day 15. The concentration of ALB in the medium was 0.2 micro g/ml on day 11, and increased to 2.2 micro g/ml by day 15. ALB-positive cells under ICC manifest morphological structures were consistent with normal mouse hepatocytes. The differentiation ratio of hepatocytes in the ES cell differentiation system was 30% on day 15 (significantly lower without the presence of growth factors). CONCLUSIONS: ES cells can differentiate into mature hepatocytes. Growth factors, such as aFGF and HGF, can enhance this differentiation and produce sufficient numbers of functional hepatocytes. This method may be a reliable new way of differentiating ES cells into hepatocytes for use in replacement therapy in the treatment of liver failure.

Effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD_(34)^+ hematopoietic cells in bone marrow of asthmatic mice

BACKGROUND: Corticosteroids remain the most effective therapy available for asthma. They have widespread effects on asthmatic airway inflammation. However, little is known about the effects of corticosteroids on the production of bone marrow inflammatory cells in asthma. This study observed the effects of glucocorticoid and cysteinyl leukotriene 1 receptor antagonist on CD34+ hematopoietic cells, so as to explore the possible effectiveness of a bone marrow-targeted anti-inflammatory strategy. METHODS: Balb/c mice were sensitized and challenged with ovalbumin (OVA) to establish an asthmatic model. For two consecutive weeks, asthmatic mice were challenged with OVA while being given either prednisone, montelukast, prednisone plus montelukast, or sterile saline solution. The mice were killed 24 hours after the last challenge with OVA, and bronchoalveolar lavage fluid (BALF), peripheral blood, and bone marrow were collected. Eosinophils in peripheral blood and BALF, and nucleated cells in BALF, peripheral blood, and bone marrow were counted. The percentages of CD34+ cells, CD4+ T lymphocytes and CD8+ T lymphocytes among nucleated cells in peripheral blood and bone marrow were counted by flow cytometry. Immunocytochemistry and in situ hybridization were employed to detect expression of CD34 and interleukin (IL)-5Ralpha mRNA (CD34+ IL-5Ralpha mRNA+ cells) among bone marrow hematopoietic cells. RESULTS: Compared with the sterile saline solution group, the number of eosinophils in BALF and peripheral blood, CD34+ cells in peripheral blood and bone marrow, and CD34+ IL-5Ralpha mRNA+ cells in bone marrow of mice from the prednisone and prednisone plus montelukast groups were significantly lower (P

Relationship between bone marrow-derived CD_(34)^+ cells expressing interleukin-5 receptor messenger RNA and asthmatic airway inflammation

BACKGROUND: Asthma is clinically related with the degree of eosinophilic inflammation. How asthmatic airway inflammation is affected is still poorly understood. So the effects of bone marrow-derived hematopoietic cells expressing CD(34) (CD(34)(+)) and interleukin-5 (IL-5) receptor messenger RNA (IL-5R mRNA+) on asthmatic airway inflammation were investigated. METHODS: Balb/c mice were sensitized and challenged by ovalbumin (OVA) to establish an asthmatic model while control mice were sensitized and exposed to sterile saline. The mice were killed at different time points after being challenged by OVA and sterile saline. Then, bronchoalveolar lavage fluid (BALF), peripheral blood (PB) and bone marrow (BM) were prepared. Eosinophils in PB (PBEOS) and BALF (BALFEOS), nuclear cells in BALF, PB and BM were counted. By flow cytometry, the percentage of CD(34)(+) cells to nucleated cells in PB, BM and the relative number of CD(34)(+) cells in PB (PBCD(34)(+)) and BM (BMCD(34)(+)) were calculated. Immunocytochemistry and in situ hybridization were used to investigate the hematopoietic cells with co-localized expression of CD(34) and IL-5R mRNA in BM (BMCD34+IL-5R mRNA+). The percentage of BMCD34+IL-5R mRNA+ to BMCD(34)(+) was calculated. RESULTS: Twelve hours after challenge by OVA, BALFEOS and PBEOS in the experimental group were significantly higher than those in the control group (P